3D C(CC)H TOCSY experiment for assigning protons and carbons in uniformly 13C- and selectively 2H-labeled RNA.
We have prepared RNAs uniformly 13C-labeled in the ribose ring and with the H3',H4',H5'/H5" protons specifically replaced with deuterons (3',4',5'/5"-2H-13C, termed d4-13C ribose), which offers the advantage of spectral simplification without sacrifice of sensitivity for the remaining protons. A 3D C(CC)H TOCSY experiment that uses a combination of cross-polarization transfer and deuterium decoupling improves the transfer of magnetization around the ribose ring and facilitates assignment of all ribose carbons and H1' and H2' protons in a 30-nucleotide RNA from HIV-2. These and other combinations of labeling and pulse sequence methodology should prove invaluable for the study of large RNAs, RNA-ligand, and RNA-protein complexes.
Dayie
K T
KT
Chemistry Department, Massachusetts Institute of Technology, Cambridge 02139, USA.
Tolbert
T J
TJ
Williamson
J R
JR
D002247:Carbon Isotopes; D003903:Deuterium; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D012313:RNA / Q000737:chemistry*; D012266:Ribose / Q000737:chemistry
D002247:Carbon Isotopes; D008024:Ligands; D012313:RNA; D012266:Ribose; D003903:Deuterium